The present invention relates to anti-Galxcex1(1,3)Gal antibody binding peptides and to the use of such peptides in cancer therapy.
A successful vaccine for cancer immunotherapy requires the identification of a suitable target antigen and the production of a cytotoxic T cell response (25). Cancer mucins, particularly MUC1 (2), provide a suitable target in cancer as there is a 10-fold increase in mucin expression, a ubiquitous (rather than polar) distribution on the cell surface, and altered glycosylation which reveals normally hidden peptide sequences (particularly an anti-MUC1 antibody detected epitope: the amino acids APDTR from the variable number of tandem repeat region (VNTR) (27). These changes apparently absent in normal mucin generate new targets for immunotherapy (28). The APDTR sequence is immunogenic in mice leading to antibody formation whether the antigen is administered as purified mucin (MHFG) or peptides (29). Such studies of immunogenicity in mice would be of little relevance to humans, were it not for the findings that tumour specific CTLp exist in the lymph nodes of patients with cancers of either breast, ovary or pancreas (30). Thus, theoretically, patients could be immunised with MUC1 peptide sequences to convert their CTLp into functional CTLs which should have a therapeutic anti-cancer effect.
The applicants have previously shown in a murine MUC1+ tumour model, that a 20 mer MUC1 VNTR peptide sequence (made as a GST fusion protein (FP)) when coupled to oxidised mannan (M-FP-oxidised) generates H-2 restricted CTLs which protects from challenge with MUC1+ mouse tumours, and in addition leads to the rapid reversal of the growth of established MUC1+ tumours (stimulation of T1 T cells) (International Patent Application No. PCT/AU9400789) (31,32). Based on the foregoing, adenocarcinoma patients have been immunised with M-FP and antibody and cellular responses generated. Nonetheless it is of interest that patients could be immunised, albeit weakly, against a self peptide, and both T and B cell tolerance appears to be broken. Despite the immune responses noted, MUC1 is a self peptide occurring in normal mucin in tissues such as breast, kidney, lung, ovary. As it is ubiquitous, anti-mucin responses have the potential for inducing autoimmune diseases against any of the normal tissues.
Surprisingly, when conducting investigations with peptides developed to bind with anti-Galxcex1(1,3)Gal antibodies in relation to the problem of hyperacute xenograft rejection, it was found by the present inventors that the human mucin peptides (arising from muc1-muc4 genes, listed as muc pep 1-11 in table 1) also bound to the anti-Galxcex1(1,3)Gal antibody (see co-pending international patent application filed on Sep. 27, 1996, also based on Australian Patent Application No. PN 5680/95). Given that the peptides developed to bind to anti-Galxcex1(1,3)Gal antibodies exhibit similar antibody binding characteristics to the human mucin peptides, it was postulated that such peptides could be useful in generating an immune response against tumour antigens. Being non-self peptides this immune response has the potential to be greater than that generated by self peptides such as MUC1.
Accordingly therefore, it is an object of the present invention to develop novel cancer immunotherapy vaccines and methods of cancer therapy. Other objects of the present invention will become apparent from the following description thereof.
According to one embodiment of the present invention there is provided a cancer vaccine comprising a peptide which mimicks MUC1 or other cancer peptides and one or more pharmaceutically acceptable carrier or diluent, optionally in association with an appropriate carrier peptide or another therapeutic agent.
According to another embodiment of the present invention there is provided a method of treatment of a human patient suffering from or prone to suffer from cancer, which comprises administering to said patient an effective amount of a cancer vaccine comprising a peptide which mimicks MUC1 or other cancer peptides, and optionally one or more pharmaceutically acceptable carrier or diluent, optionally also in association with one or more appropriate carrier peptides or another therapeutic agents.
It is to be recognised that throughout this specification, unless the context requires otherwise, the word xe2x80x9ccomprisexe2x80x9d, or variations thereof such as xe2x80x9ccomprisesxe2x80x9d or xe2x80x9ccomprisingxe2x80x9d, will be understood to imply the inclusion of a stated element or integer or a group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.
It was recognised by the inventors of the present application that xcex1-galactosyl sugars such as d-galactose, melibiose, stachyose, methyl-xcex1-d-galactopyranoside, D-galactosamine and derivatives thereof bind to anti-Galxcex1(1,3)Gal antibodies. It was also noted by the present inventors that the Galxcex1(1,3)Gal epitope is present on the surface of endothelial cells of animals other than higher primates (humans and old world monkeys). Higher primates do not make Galxcex1(1,3)Gal as they lack a functional xcex1(1,3) galactosyl transferase due to the presence of multiple in-phase stop codons and multiple insertions and deletions leading to frame shifts within the xcex1(1,3)galactosyl transferase genes, which result in non-functional pseudo-genes (8,7). All humans however, have natural antibodies to Galxcex1(1,3)Gal(15,18), which is probably due to immiunisation with bacteria which carry xcex1-linked Gal as part of the lipopolysaccharide.
It is also known that the IB4 lectin which is a plant glycoprotein from the species Griffonia simplicifolia, is capable of binding to the Galxcex1(1,3)Gal epitope. On this basis therefore, the present inventors proposed to locate peptides capable of binding to the anti-Galxcex1(1,3)Gal antibody by screening peptides for binding with the IB4 lectin. A multitude of random amino acid sequence octapeptides was synthesised and displayed in a phage display library and screened for binding activity against the IB4 lectin, the surprising result of which was that a number of synthesised peptides demonstrated binding with the IB4 lectin. Similarly, other known peptides were also screened for binding against the IB4 lectin and it was surprisingly also found that the IB4 lectin bound to the protein core of the human mucin peptides (MUC 1-7) which are highly expressed on the surface of tumour cells (2,19) and which in this situation have an altered pattern of glycosylation which leads to exposure of the protein core (3). It has since been demonstrated by the present inventors that the IB4 binding synthetic peptides (Gal pep 1 to Gal pep 7) obtained from the random peptide library and the mucin peptides both bind to the anti-Galxcex1(1,3)Gal antibody, and can be utilised in cancer therapy protocols.
The phrase within the specification peptides xe2x80x9cmimicks MUC1 or other cancer peptidesxe2x80x9d encompasses all peptides which demonstrate analogous binding in the groove of class I MHC molecules. An indicator of mimicking MUC1 is binding to IB4 or anti-Galxcex1(1,3)Gal antibodies. Relative affinities of peptides for the anti-Galxcex1(1,3)Gal antibody or for IB4 can be calculated by comparing the molar concentration required to obtain 50% inhibition of the binding of antibody by lectin or sugars, using an ELISA (enzyrme-linked immunosorbent assay). The details of this technique will be described more fully in the examples under the heading xe2x80x9cmaterials and methodsxe2x80x9d. The peptides of the invention include peptides having only as few as 3 amino acids up to polypeptides including up to or exceeding 200 amino acids. Although, as will be discussed in further detail, preferred peptides of the invention include the consensus is sequence ArXXArZ (as defined below) and the peptides Gal pep 1 to Gal pep 7 (SEQ ID Nos. 1-7, respectably), this is in no way to be considered limiting upon the invention. Other peptides which demonstrate the requisite cancer peptide mimicking, and particularly peptides which are similar to Gal pep 1-Gal pep 7 (SEQ ID Nos. 1-7, respectably) but which include one or more deletions or modifications to the amino acid sequence are to be considered to fall within the scope of the invention.
Although not to be considered limiting upon the scope of the invention, it is noted that the peptides exhibiting antibody binding characteristics which were located from the random amino acid library screening have the following consensus sequence:
ArXXArZ
where Ar=tryptophan (W), phenylalanine (F) or tyrosine (Y)
X=a small aliphatic or polar amino acid residue such as for example glutamic acid O), serine (S) aspartic acid (D), glycine (G), isoleucine (I), alanine (A) or asparagine (N).
Z=branched aliphatic amino acid such as for example valine (V) isoleucine (I), or leucine (L).
Particularly preferred are the peptides shown in FIG. 1 as Gal pep 1 to Gal pep 7 (SEQ ID Nos. 1-7, respectably) which each conform with the above consensus sequence.
Other preferred compounds are the mucin glycopeptides MUC 1 to MUC 7 (4).
The most preferred peptide however, is Gal pep 1 which has the sequence DAHWESWL (SEQ ID No. 1), and which can mimic the conformation of the MUC1 VNTR peptides SAPDTRPAP/APDTRPAPG (SEQ ID No. 9/SEQ ID No. 10) (which bind H-2Dd and H-2Ld respectively).
The applicants have previously shown that mice immunised with mannan-MUC1-peptides make cytotoxic T cells (CTLs), little antibody and are protected from MUC1+ tumour growth. The same specific anti-MUC1 responses can be produced by immunising with the peptides of the invention and particularly the DAHWESWL peptide (SEQ ID No. 1) linked to KLH, in that aniti-MUC1 (and anti-DAHWESWL) CTL responses can be induced or antibody produced and more particularly, specific tumour protection occurs of magnitude greater than or similar to that obtained with mannan-MUC1 peptide immunisation.
It is also possible that the peptides as outlined above which accord with the present invention can be conjugated to other species. The other species comprehended include all chemical species which can be fused to the peptide in question without affecting the binding of the peptide by T-cells. Specific examples are for example other antigens which may elicit a separate immune response, carrier molecules which may aid absorption or protect the peptide concerned from enzyme action in order to improve the effective half life of the peptide. Other possibilities are conjugation of peptides to solid or liquid phases for use in immuno assays for diagnostic or therapeutic purposes. Specific examples of peptide conjugation are conjugation to other serum proteins or macromolecules.
The present invention relates to methods of cancer therapy which involve the peptides according to the present invention. In particular vaccines which include peptides according to the invention can be administered to cancer patients to induce immunity to the mucin core of aberrantly glycosylated mucins which are highly expressed on the surface of tumour cells. Mucins are expressed in adenocarcinomas in cancer of the breast, liver, colon, prostate and others. In treating cancer it may useful to conjugate peptides to other carriers to induce T cells capable of recognising and destroying cancer cells.
The present invention also relates to methods of vaccinating human subjects as a method of cancer therapy or treatment for auto-immune disease. In this way the inventive vaccine can be administered to human patients who are either suffering from, or prone to suffer from cancer or autoimmune disease.
In cancer therapy it is possible to immunise with peptide-carrier combination to induce T cells capable of recognising and destroying target cancer cells or alternatively immunisation to induce antibodies with anti-tumour activity.
The vaccine according to the invention may contain a single peptide according to the invention or a range of peptides which cover different or similar pepitopes. In addition or alternatively, a single polypeptide may be provided with multiple epitopes. The latter type of vaccine is referred to as a polyvalent vaccine.
In a preferred embodiment of the invention the peptide is conjugated to a carrier protein such as for example tetanus toxoid, diphtheria toxoid or oxidised KLH in order to stimulate T cell help.
The formation of vaccines is generally known in the art and reference can conveniently be made to Remington""s Pharmaceutical Sciences, 17th ed., Mack Publishing Co., Easton, Pa., USA.
For example, from about 0.05 ug to about 20 mg per kilogram of body weight per day may be administered. Dosage regime may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. The active compound may be administered in a convenient manner such as by the oral, intravenous (where water soluble), intramuscular, subcutaneous, intra nasal, intradermal or suppository routes or implanting (eg using slow release molecules by the intraperitoneal route or by using cells e.g. monocytes or dendrite cells sensitised in vitro and adoptively transferred to the recipient). Depending on the route of administration, the peptide may be required to be coated in a material to protect it from the action of enzymes, acids and other natural conditions which may inactivate said ingredients.
For example, the low lipophilicity of the peptides will allow them to be destroyed in the gastrointestinal tract by enzymes capable of cleaving peptide bonds and in the stomach by acid hydrolysis. In order to administer peptides by other than parenteral administration, they will be coated by, or administered with, a material to prevent its inactivation. For example, peptides may be administered in an adjuvant, co-administered with enzyme inhibitors or in liposomes. Adjuvants contemplated herein include resorcinols, non-ionic surfactants such as polyoxyethylene oleyl ether and n-hexadecyl polyethylene ether. Enzyme inhibitors include pancreatic trypsin inhibitor, diisopropylfluorophosphate (DEP) and trasylol. Liposomes include water-in-oil-in-water CGF emulsions as well as conventional liposomes.
The active compounds may also be administered parenterally or intraperitoneally. Dispersions can also be prepared in glycerol liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
The pharmaceutical forms suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of superfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, chlorobutanol, phenol, sorbic acid, theomersal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the composition of agents delaying absorption, for example, aluminium monostearate and gelatin.
Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in th e appropriate solvent with various of the other ingr edients enumerated above, as required, followed by filtered sterliation. Generally, dispersions are prepared by incorporating the various sterided active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the p reparation of sterile injectable solutions, t he preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
When the peptides are suitably protected as described above, the active compound may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet. For oral therapeutic administration, the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, waftes, and the like. Such compositions and preparations should contain at least 1% by weight of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 5 to about 80% of the weight of the unit. The amount of active compound in such therapeutically useful compositions is such that a suitable dosage will be obtained. Preferred compositions or preparations according to the present invention are prepared so that an oral dosage unit form contains between about 0.1 ug and 2000 mg of active compound.
The tablets, pills, capsules and the like may also contain the following: A binder such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose or saccharin may be added or a flavouring agent such as peppermint, oil of wintergreen, or cherry flavouring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavouring such as cherry or orange flavour. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compound may be incorporated into sustained-release preparations and formulations.
As used herein xe2x80x9cpharmaceutically acceptable carrier and/or diluentxe2x80x9d includes any and all solvents, dispersion media, coatings antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the novel dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active material and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active material for the treatment of disease in living subjects having a diseased condition in which bodily health is impaired as herein disclosed in detail.
The principal active ingredient is compounded for convenient and effective administration in effective amounts with a suitable pharmaceutically acceptable carrier in dosage unit form as hereinbefore disclosed. A unit dosage form can, for example, contain the principal active compound in amounts ranging from 0.5 xcexcg to about 2000 mg. Expressed in proportions, the active compound is generally present in from about 0.5 xcexcg/ml of carrier. In the case of compositions containing supplementary active ingredients, the dosages are determined by reference to the usual dose and manner of administration of the said ingredients.
Still another aspect of the present invention is directed to antibodies to the peptides. Such antibodies may be monoclonal or polyclonal and may be selected from naturally occurring antibodies to the peptide or may be specifically raised to the peptides. In the case of the latter, the peptides may need first to be associated with a carrier molecule. The antibodies and/or peptides of the present invention are particularly useful for immunotherapy and vaccination and may also be used as a diagnostic tool for infection or for monitoring the progress of a vaccination or therapeutic regime.
In another aspect of the invention there are provided nucleotide sequences encoding the proteins according to the present invention. Preferably the nucleotide sequence encodes Gal pep 1.
Nucleotide sequences may be in the form of DNA, RNA or mixtures thereof. Nucleotide sequences or isolated nucleic acids may be inserted into replicating DNA, RNA or DNA/RNA vectors as are well known in the art, such as plasmids, viral vectors, and the like (Sambrook et al, Molecular Cloning A Laboratory Manual, Coldspring Harbour Laboratory Press, NY, second edition 1989). Nucleotide sequences encoding the antibodies of the present invention may include promoters, enhances and other regulatory sequences necessary for expression, transcription and translation. Vectors encoding such sequences may include restriction enzyme sites for the insertion of additional genes and/or selection markers, as well as elements necessary for propagation and maintenance of vectors within cells.
Nucleotide sequences encoding antibodies according to the present invention may be used in homologous recombination techniques as are well known in the art Capecchi M R, Altering the Gene by Homologous Recombination, Science 244:1288-1292, 1989; Merleno G T, Transgenic Animals in Biomedical Research, FASEB J 5:2996-3-001, 1992; Cosgrove et al, Mice Lacking MAHC class II molecules, cell 66:1051-1066, 1991; Zijlstra et al, Germ-Line Transmission of a Disrupted B2-Microglobulin Gene Produced by Homologous Recombination in Embryonic Stem Cells, Nature 342:435, 1989). In such techniques, nucleotide sequences encoding the peptides according to the invention are recombined with genomic sequences in stem cells, ova or newly fertilised cells comprising from 1 to about 500 cells. Nucleotide sequences utilised in homologous recombination may be in the form of isolated nucleic sequences or in the context of vectors. Insertion of new active genes by transgenesis is also comprehended.